1. Field of the Invention
The present invention relates to live attenuated bacteria for use in vaccines.
2. Description of the Related Art
Shiga toxin-producing strains of Escherichia coli (STEC), also known as enterohemorrhagic Escherichia coli (EHEC) are important foodborne pathogens associated with foodborne epidemics of bloody diarrhea and hemorrhagic colitis (HC) (Nataro et al., 1998). While HC is often self-limiting, STEC infection can lead to more severe complications including central nervous system (CNS) disturbance and fatal hemolytic uremic syndrome (HUS) (Karmali et al., 1985). HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure and is especially life-threatening for the young and elderly (Carter et al., 1987 and Nataro et al., 1998). Human disease-associated STEC strains are referred as enterohemorrhagic E. coli (EHEC). According to the Center for Disease Control (CDC), there are about 73,000 cases of STEC infections in the U.S. each year. HUS occurs in 5-10% of these cases and leads to as many as 250 deaths (Boyce et al., 1995; Nataro et al., 1998). In the U.S., EHEC are most often serotype O157:H7, but strains in other serogroups including 026, 0111 also cause disease. Although STEC strains are generally susceptible to a variety of antibiotics, there are retrospective studies showing that the use of antibiotics negatively alters the outcome of STEC infections leading to increased incidence of HUS (Nataro et al., 1998). This is likely because lysis of bacteria by some antibiotics leads to increased release of toxin, as well as to increased toxin synthesis during the induction of lysogenic toxin-producing bacteriophage. Second, antibiotic therapy may alter the balance of intestinal flora thereby increasing the systemic absorption of released toxin (Nataro et al., 1998). Infections caused by STEC have became a public health concern since outbreaks of the disease were reported following ingestion of undercooked ground beef in hamburgers distributed by national fast food chains. Outbreaks of infection with STEC are likely to continue because of the capacity for wide distribution of the infecting organism provided by an efficient food distribution system. Currently, there are no proven vaccines or therapeutic agents for infections caused by STEC (EHEC).
Cattle are most frequently identified as the primary source of EHEC infection. EHEC thrives in the ruminant gastrointestinal tract, farm water troughs, raw manure, and other contaminated environmental surfaces (Hovde et al., 1999). The bacteria can survive for more than 2 to 5 months in water containing rumen content (Doyle, 2003 and Killham et al., 2003). The EHEC organisms may be shed at levels up to 106 cfu per gram of feces for several weeks following infection (Naylor et al., 2003 and Omisakin et al., 2003). Although natural symptomatic STEC infections have been reported in young calves (Janke et al., 1989; Person et al., 1989; Pospischil et al., 1987; Schoonderwoerd et al., 1988; and Wray et al., 1989), colonization in adult cattle by EHEC causes no clinical disease. EHEC isolates from cattle produce Shiga toxin (both Stx-1 and/or Stx-2), however, the mechanisms for resistance of adult cattle to STEC disease, despite documented intestinal carriage, is still unknown (Pospischil et al., 1987; and Stordeur et al., 2000). It is important to recognize that the rectum and cecum are principal sites of STEC 0157:H7 colonization during the carrier-shedder state in cattle (Dean-Nystrom, 2003).
The broad outline of the pathogenic mechanisms of STEC infections in humans, due to strain 0157:H7 and other STEC, are well known. After ingestion of contaminated food or water, STEC colonize the intestine (primarily the large bowel), utilizing a mechanism of intimate adherence to intestinal epithelial cells, and elaborate a potent toxin, Shiga toxin (Stx), which is the major virulence factor of this organism (Nataro et al., 1998). Locally produced Stx is then absorbed into the circulation and targets microvascular endothelial cells containing specific receptors for Stx. Low levels of Stx reaching the circulation are able to induce profound vascular lesions in target organs including bowel, central nervous system and kidney (Gyles, 1994).
The hallmark of pathogenicity of EHEC is the production of Stx implicated in the development of HUS (Griffin et al., 1991; Kaper, 1998; Noel et al., 1997; and O'Brien et al., 1992). Stx(s) produced by EHEC belong to a family of bacterial cytotoxins structurally related to those produced by the dysentery bacillus Shigella dysenteriae (Tesh et al., 1991; and O'Brien et al., 1992). Both Stx-1 and Stx-2 are in the class known as AB toxins composed of one A subunit and five identical receptor-binding B subunits (Jackson, 1990,—and Jackson et al., 1990). The B subunit binds to a receptor molecule on the host cell surface (Jackson, 1990; Jackson et al., 1990; and Mobassaleh et al., 1988). The A subunits of both toxins are highly selective N-glycosidases that depurinate a specific adenine residue on the eukaryotic 60S ribosomal subunit thus blocking protein synthesis and leading to the death of the cell (Hovde et al., 1988; and Jackson et al., 1990). Shiga toxins can modulate cytokine secretion and function. For instance, Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells, and their N-glycosidase activity is essential for the induction because proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 but not by a non-toxic mutant of Stx1 which lacks N-glycosidase activity (Yamasaki et al., 1999). Microarray analysis demonstrated upregulation of genes belonging to chemokines and cytokines and other genes encoding cell adhesion molecules and transcription factors that are involved in immune response or apoptosis (Matussek et al., 2003).
Attaching and effacing Escherichia coli (AEEC) represent a group of enteric pathogens of humans and animals, including human hEPEC, a major cause of infant diarrhea; EHEC, an important food-borne pathogen; strains causing diarrhea in animals such as rabbit (rEPEC), pig and dog EPEC; and Citrobacter rodentium in mice (Nataro et al., 1998; An et al., 1997; Cantey et al., 1977; and Zhu et al., 1994). Central to the pathogenesis of AEEC infection is the formation of attaching/effacing (A/E) lesions characterized by intimate bacterial attachment to intestinal epithelial cells and effacement of microvilli with disruption of host cell cytoskeleton (Nataro et al., 1998). The genes essential for the A/E phenotype are encoded on the locus of enterocyte effacement (LEE) pathogenicity island (PAI), the complete nucleotide sequence of which has been obtained from hEPEC O127:H6 (strain E2348/69) (Elliott et al., 1998), EHEC O157:H7 (strain EDL933) (Perna et al., 1998), rEPEC strain RDEC-I (015:H-) (Zhu et al., 2001), and C. rodentium associated with colonic hyperplasia in mice (Deng et al., 2001).
LEEs of EPEC, EHEC, rEPEC, and C. rodentium share a 34-kb conserved region containing 40 (RDEC-I) or 41 (hEPEC, EHEC or C. rodentium) open reading frames (ORF) organized into five major polycistronic operons: LEE1, LEE2, LEE3, LEE5 and LEE4, and several minor operons and monocistronic genes (Elliott et al., 1998). The conserved 34-kb core region of LEE PAIs of rEPEC, hEPEC, EHEC, and C. rodentium exhibit nearly identical genetic organization and high homology of LEE-encoded genes (FIG. 1; Elliott et al., 1998; Perna et al., 1998; Zhu et al., 2001; and Deng et al., 2001). The LEE encodes a global regulator Ler (LEE encoded regulator), a type III secretion system (TTSS) (LEE1, LEE2, LEE3), a bacterial adhesin named intimin, a translocated intimin receptor Tir and CesT chaperone for Tir (LEE 5), and several secreted effector proteins (LEE4) including Esp D, B, and F which are delivered into host cells via the TTSS (Elliott et al., 1998; Kaper et al., 2004 and Kenny et al., 1997). TTSSs are critical for the virulence of A/E organisms. While TTSS apparatus deliver LEE-encoded effector molecules, such as Tir, Map, EspF EspG, and EspH, the TTSSs also contribute to delivering virulence factors encoded outside the LEE, such as Cif, and EspF(u) (McNamara et al., 2001; and Marches et al., 2003).
Ler is encoded as the first open reading frame (ORF) in LEE1. It is highly conserved (95-98% amino acid identity) among hEPEC, EHEC and rEPEC 015:H-strain RDEC-I (Elliott et al., 1998; Perna et al., 1998; and Zhu et al., 2001). The deduced amino acid (AA) sequences of Ler from hEPEC share substantial similarities (24% identity and 44% similarity) with H—NS, the histone-like non-structural protein) of Salmonella mainly in the carboxyl terminus (Elliott et al., 2000). It has been shown that Ler plays a central role in the regulation of LEE-encoded gene expression (FIG. 2) and, in EPEC, that Ler positively regulates the LEE operons by acting as an antirepressor protein that overcomes the H—NS-mediated silencing of the LEE2/LEE3, LEE5 and LEE4 (Bustamante et al., 2001; Haack et al., 2003; and Sanchez-Sanmartin et al., 2001) Ler also activates the expression of the genes outside the LEE, such as espC encoded on a second PAI of EPEC (Elliott et al., 2000).
rEPEC constitutes a subset of the AEEC pathotype and strains of different serotypes have been shown to be causative agents of rabbit enteritis (Camguilhem et al., 1989; and Peeters et al., 1988). rEPEC induce A/E lesions in a manner similar to hEPEC and EHEC (Cantey et al., 1977). The extensive phenotypic and genotypic homologies among human and animal A/E strains suggest a common evolutionary origin and perhaps common regulatory mechanisms for LEE-encoded gene expression. A previous study demonstrated in hEPEC that the Ler is essential for in vitro pathogenic effects suggesting that a deletion mutation in the ler gene might attenuate the in vivo virulence of rEPEC (Mellies et al., 1999; and Sperandio et al., 2000).
Bacterial intimate adherence to host epithelial cells is mediated by binding of intimin to the translocated intimin receptor (Tir), which is delivered by A/E organisms to eukaryotic cells (Frankel et al., 1996a, 1996b and 1998; Hartland et al., 1999; Hicks et al., 1998; and Kenny et al., 1997). Intimin is an outer membrane protein (OMP) adhesin that shares homology with the invasin that promotes eukaryotic cell invasion by Yersinia (Jerse et al., 1990). Currently, nearly a dozen genetically and serologically distinct intimin subtypes are reported among A/E organism (Adu-Bobie et al., 1998; Oswald et al., 2000; Ramachandran et al., 2003; and Zhang et al., 2002). All currently known intimin alleles demonstrate more homology in their amino (N)-terminal regions than in their carboxy (c)-terminal regions. Intimins of A/E E. coli (AEEC) including human EPEC O127-.H6 (Intimin-α), EHEC O157:H7 (Intimin-γ), or rEPEC 015-.H-(Intimin-β) show greater than 94% amino acid (aa) identity over the N-terminal two thirds of the molecule while showing only 55% homology over the remaining one third portion at the C-terminus (Zhu et al., 2001). The crystal structure of the C-terminal EPEC intimin fragment (residues 658-939) revealed three adjacent domains: the immunoglobulin-like (Ig) D1 (residues 658-751) and D2 (residues 752-841) and the C-type lectin-like D3 (residues 842-929) (Frankel et al., 1995; and Luo et al., 2000). The immunodominant regions have been demonstrated to be in the domains D1 and D2, as shown by reaction with intimin-specific antiserum (Adu-Bobie et al., 1998). Binding of intimin and Tir is mediated primarily by interactions between the lectin-like D3 domain of intimin and the Tir intimin-binding domain (Luo et al., 2000). Within the Tir-binding region of intimin two conserved cysteine residues (aa 860 and 937 of EPEC intimin) are involved in the formation of a disulfide loop essential for intimin function (Frankel et al., 1995; Hicks et al., 1998; and Luo et al., 2000). This disulfide loop is conserved in Yersinia invasin and all the intimin molecules (Eliott et al., 1998; Ramachandran et al., 2003; and Zhu et al., 2001). Recent studies have shown that other accessory proteins promote bacterial adherence to intestinal epithelial cells, including the Efal from EHEC 0111 (Nicholls et al., 2000) and the Efal homologue LifA from EPEC 0126:H7 (Klapproth et al., 2000), the flegellin of EHEC, and some novel fimbriae (Torres et al., 2003). However, these proteins are not directly involved in the formation of A/E lesions.
Intimin is critical for intimate bacterial adherence. Attenuation of virulence by deletion of intimin has been demonstrated for human EPEC (0127:H6) (Donnenberg et al., 1993a), human EHEC (O157:H7) (Donnenberg et al., 1993b), rEPEC (O103:H2) (Marches et al., 2000), and C. rodentium (Deng et al., 2004).
The role of intimin in in vivo virulence has been tested through isogeneic mutants deficient in expression of functional intimin. Donnenberg and Kaper created an internal 1848-bp deletion in EPEC eae gene and tested its pathogenicity in humans (Donnenberg et al., 1991). While all of the human volunteers received WT EPEC developed diarrhea, the isogeneic eae mutant caused diarrhea in 4 of 11 individuals (Donnenberg et al., 1993a). In a separate study, the isogeneic eae mutant generated by replacing the internal 1.1-kb eae DNA with a 2.9-kb DNA fragment containing a Tet marker of human EHEC 86-24 (0157:H7) was unable to colonize in experimentally inoculated piglets (Donnenberg et al., 1993b). In REPEC O103:H2, an insertion of aphT encoding Kan resistance in the eae gene (between 993 nt and 994 nt) disrupted the expression of intimin and abolished bacterial virulence when tested by experimental inoculation of its natural rabbit host (Marches et al., 2000). Anti-intimin immune responses can modulate the outcome of A/E organism infection. In another study conducted in piglets, passive immunization, achieved by allowing neonatal piglets to suck colostrums from intimin-vaccinated dams for up to 8 h before inoculating with EHEC 0157:H7, protected animals from STEC 0157:H7 colonization and intestinal damage (Dean-Nystrom et al., 2002). Using mutant E. coli heat-labile enterotoxin (LT) lacking the nick site in the A subunit as an adjuvant, intranasally administered 0157:H7 intimin induced an elevation of IgA-specific antibody in the nasal secretion and saliva of calves as well as an elevation of IgG1-specific antibody level against the intimin in the sera and colostrums of cows (Yokomizo et al., 2002). In yet another study, EHEC 0157:H7 intimin C-terminal domain was expressed in transgenic tobacco plant cells and mice immunized with the plants generated an intimin-specific mucosal immune response and exhibited a reduced duration of EHEC 0157:H7 fecal shedding (Judge et al., 2004). Interestingly, vaccination of mice with Int280α induced both type-specific protection to intimin-c organisms and to heterologous intimin types indicating that a highly conserved domain of intimin (Int388-667) including part of C-terminal fragment D1 domain may have potential to induce protection against infections by A/E organism expressing different intimin types (Ghaem-Maghami et al, 2001).
More than 50 serotypes of STEC have been isolated from stool samples of patients with hemorrhagic colitis or HUS. STEC 0157:H7 is the predominant serotype reported as the causative agent world-wide (Nataro et al., 1998). Analysis of HUS samples collected from 1987 to 1991 in the United States indicated that STEC could be implicated in 72% of cases of HUS, and STEC serotype 0157 may be implicated in 80% cases studied (Banatvala et al., 2001). However, infections due to non-0157 STEC are now increasingly recognized (Nataro et al., 1998; and Tarr et al., 1996 and 2002). Of these, STEC 026 and STEC 0111 have been isolated most frequently. In Boston and Virginia, approximately half of all Stx-producing E. coli isolates from patients were of non-0157:H7 serotypes (Park et al., 1996). STEC serogroup 026 and 0111 have been increasingly associated with outbreaks in Europe, Japan, Australia, India, where they account for the majority of HUS cases (Ojeda et al., 1995,—Pierard et al., 1990; and Robins-browne et al., 1998). The prevalence of 0157, 026, and 0111 in humans is in accordance with the findings that these serogroups were most common in fecal samples from animals (Blanco et al., 2004a and 2004b; Borie et al., 1997; and Rey et al., 2003).
Vaccines for animals are aimed at reduction of EHEC secretion in their natural host. A clinical trial of a parenteral STEC vaccine has recently been conducted by the Canadian investigators (Potter et al., 2004). The vaccine formulation containing secreted protein preparations of STEC strain 0157:H7, together with aluminum adjuvant, VSA3 was delivered subcutaneously in the necks of seronegative cattle (Potter et al., 2004). Vaccinated cattle were primed and showed an increase in serum IgG antibody titers. Vaccination was reported to reduce the prevalence of STEC 0157:H7 from 21.3% to 8.8% in feedlot cattle at the day of marketing (Potter et al., 2004). Thus, although an apparent effect was seen, substantial numbers of vaccinated animals still shed STEC in the feces.
In a non-vaccine study, the combinations of several strains of probiotics were shown to inhibit the growth of EHEC 0157 in vitro and reduce the fecal shedding of EHEC 0157:H7 (Doyle, et al, 2003). The fecal shedding and pathogenicity of STEC O26:H11, 0111:NM, 0157:H7 in weaned calves (8 to 10 weeks of age) were compared with and without treatment using a three-strain mixture (Hicks et al., 1998 and Tkalcic et al., 2003). The probiotic E. coli substantially reduced or eliminated fecal shedding of 0157:H7 and 0111:NM. However, STEC were still recovered from one third of calves receiving the probiotic treatment, and the probiotic E. coli did not reduce fecal shedding or gastrointestinal persistence of 026:HIl (Hicks et al., 1998 and Tkalcic et al., 2003). Interestingly, when probiotics were used in calves of less that 1 week of age, reduced fecal shedding of 0111:NM and O26:H11 but not STEC 0157 was observed in most calves (Zhao et al., 2003).
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